Imaging in diffusive media has become an attractive field of research mainly due to its applications in biology and medicine, showing great potential in, for example, cancer research, drug development, inflammation and molecular biology. With the development of highly specific activatable fluorescent probes, a new way of obtaining information at the molecular level in vivo has been made possible. Due to the inherent scattering present in tissues in the optical wavelengths, light is multiply scattered in tissue and its original direction of propagation is randomized after what is termed the scattering mean free path, lsc, a distance which represents the density and efficiency of scattering of the different constituents of tissue. Even though images of fluorescent or bioluminescent emission from tissues might be recorded, due to the scattering present, there is a non-linear relation of these images with the concentration, size and position of the fluorescent probes or bioluminescent reporters, resulting in what is termed an ill-posed problem. Recently developed methods and systems make use of tomographic approaches by introducing a spatial dependence of fluorescence on the excitation and appropriate modeling of light propagation in tissues through the diffusion approximation to alleviate this ill-posedness, enabling the recovery of the spatial distribution of the concentration of fluorescence with a resolution on the order of the scattering mean free path (lsc) or better. In an ideal situation, the sensitivity of these tomographic techniques would depend only on the detector efficiency and probe brightness together with the absorption present in tissue, assuming it is possible to completely block the excitation light when measuring fluorescence. In this ideal case, the sensitivity could be increased by augmenting the laser power since the emitted and excitation intensities are proportional to one another, at least for powers acceptable for small animal imaging, which ensure that no appreciable heating occurs in tissue.
However, the reality of an in vivo measurement is quite different. Due to the excitation of surrounding auto-fluorescence always present in tissue, the sensitivity of a system is strongly dependent on the ability to distinguish the specific signal due to the fluorescent probe from the non-specific signal of the surrounding auto-fluorescence. This problem becomes more pronounced when exciting in reflection mode, since the greater auto-fluorescent contribution would be from the tissue sections closest to the camera. In practice, the sensitivity of the tomographic data, or in general any collection of fluorescent data, is determined by the level of auto-fluorescence when compared to the specific signal. This issue is overcome to some extent by using far-red or near infra-red (NIR) fluorescence signals, since tissue auto-fluorescence is slightly reduced in this part of the spectrum, with the added advantage that tissue presents lower absorption properties in this part of the spectrum. This is the case when using fluorescent probes that emit in the far-red or near infra-red part of the spectrum, which are activated when a specific molecular activity is present. An advantage of this kind of probes is that they provide a high signal-to-noise ratio. However, in most practical instances, the amount of signal that can be detected depends on how much specific signal surpasses the surrounding tissue auto-fluorescence.
In order to separate the contribution of the specific from the non-specific signal, the most common approach is to employ multi-spectral measurements, assuming the emission spectrum is known. Even though this approach slightly increases the sensitivity, the problem still remains: at each emission wavelength measured there is an unknown contribution from tissue auto-fluorescence. The weaker the specific signal, the more dominant the effect of auto-fluorescence.
Bioluminescent reporters offer the significant advantage of not requiring an external illumination to place them in an excited state such that they would emit light. Since the excited state is reached though a chemical reaction, the emitted light represents the background-free solution of the imaging problem, akin to the ideal case of fluorescence mentioned in the previous paragraphs. This inherent benefit of bioluminescence also has some drawbacks, specifically in relation to the ill-posed problem mentioned above. Since it is not currently possible to effectively introduce a spatial dependence on the intensity of this emission (whereas this is possible in the case of fluorescence), it is not possible to recover simultaneously the spatial distribution of bioluminescent probe concentration. Thus, because the main goal is to recover the spatial distribution of probe concentration, the ill-posedness of the problem cannot be substantially reduced in the case of bioluminescence as is possible in the case of fluorescence.
There is a need for an optical imaging system in which the low-background and lack of auto-fluorescence of bioluminescent probes can be combined with the specificity, high quantum yield, and the external capability of emission intensity modulation exhibited by fluorescent probes.